AvanViva is developing DART technology, a novel protein display technology, to generate leads for pharmaceutics. This SBIR application is focused on generating a high diversity library of DARTs, where each DART contains a displayed peptide directly linked to the DNA that encodes it. DART libraries could be subjected to directed evolution selections to generate and optimize leads for therapeutic antibodies or peptide pharmaceutics. AvanViva plans to leverage results from this research program to generate and commercialize leads for new peptide pharmaceutics to treat cancer and other major diseases. Background. Directed evolution is a process whereby a diverse population of proteins or peptides is screened for ones with desired properties, such as the ability to bind a drug target. Phage display and other display technologies are powerful directed evolution technologies, but they have had limited success in generating drug leads against G-protein coupled receptors (GPCRs), historically the most commercially important class of drug targets. AvanViva's DART technology may provide a powerful new solution to this challenge. A DART contains a peptide or protein drug candidate covalently linked to the DNA that encodes it using the VirD2 linker protein. Cell-free coupled transcription and translation reactions (Txn/TIn) might be used to generate high diversity libraries of DARTs in a single cell-free reaction, which could then be subjected to directed evolution selections to generate peptide or antibody drug leads. Research program. The specific aims of this study are to: 1 - Verify that the VirD2 linker protein is active in Txn/TIn; 2 - Synthesize a DART using Txn/TIn and purify it; 3 - Mix two different DART species together and purify one away from the other; 4 - Mix DNA templates encoding two different DART species in a single Txn/TIn reaction and purify one of the resulting DART species; 5 - Generate a library of peptide-bearing DARTs in a single Txn/TIn and purify one of the resulting DART species. Commercial impact. Synthesizing a high diversity library of DARTs is the last key step in laying the groundwork for DART directed evolution. Therefore, this SBIR research program would enable AvanViva to perform directed evolution to generate and optimize leads for peptide or antibody pharmaceutics, directed against GPCRs, to treat cancer and other major diseases. [unreadable] [unreadable]